ORIGINAL PAPER

Characterization of Phenytoin, Carbamazepine, Vinpocetineand Clorgyline Simultaneous Effects on Sodium Channelsand Catecholamine Metabolism in Rat Striatal Nerve Endings

Marıa Sitges Æ Blanca I. Aldana Æ Luz M. Chiu ÆVladimir Nekrassov

Accepted: 7 July 2008 / Published online: 19 August 2008

� Springer Science+Business Media, LLC 2008

Abstract The effects of two classic antiepileptic drugs

(carbamazepine and phenytoin), a potential antiepileptic

(vinpocetine) and a monoamine-oxidase inhibitor (clorgy-

line) on the simultaneous changes (detected by HPLC) on

Glu, Asp, dopamine and DOPAC inside and outside striatal

isolated nerve endings were investigated. Under resting

conditions phenytoin, carbamazepine and clorgyline

increased dopamine release. Phenytoin and clorgyline

increased internal dopamine and decreased DOPAC for-

mation. Carbamazepine decreased internal dopamine and

practically did not change DOPAC formation. Glu and Asp

release was unchanged. Neurotransmitter release induced

by the Na+ channel opener veratridine was reduced by all

the antiepileptic drugs tested, except phenytoin which, like

clorgyline, facilitated veratridine-induced dopamine

release. We conclude that besides the antagonism exerted

by carbamazepine, phenytoin and vinpocetine on excitatory

neurotransmitters release triggered by Na+ channel acti-

vation, that might importantly contribute to their

anticonvulsant action, they exert different actions on stri-

atal dopamine distribution, that might explain their

different side effect profiles.

Keywords Antiepileptic drugs � Monoamino-oxidase �Dopamine � DOPAC � Glutamate � Aspartate � Veratridine

Introduction

Several of the most effective antiepileptic drugs are

believed to stop the paroxysmal neuronal activity acting as

sodium channel blockers. For instance, the involvement of

voltage-gated sodium channels in the mechanism of action

of the anticonvulsants, carbamazepine and phenytoin, was

indicated by the inhibition of the sustained repetitive firing

in spinal cord neurons and neurons in culture from the

seminal work of Macdonald and McLean [1].

Veratridine is a toxin of natural origin that binds to Na+

channel site 2 (voltage sensor) impeding its inactivation

and by this mean increases the rate of Na+ entry. The

increase in the internal Na+ concentration induced by

veratridine in cerebral isolated nerve endings (synapto-

somes) preloaded with the Na+ selective indicator dye,

SBFI is inhibited by the Na+ channel blocker tetrodotoxin

and independent of external Ca2+ [2]. Also neurotrans-

mitter release induced by veratridine in synaptosomes is

sensitive to tetrodotoxin, is independent of external Ca2+

and depends on the presence of external Na+ [3–5].

Therefore sensitivity of veratridine-induced responses to a

drug usually indicates Na+ channel antagonism.

In hippocampal synaptosomes preloaded with radioac-

tive Glu ([3H]Glu) several of the most amply used

antiepileptic drugs and the new potential antiepileptic

vinpocetine (ethyl apovincamine-22-oate) inhibited

[3H]Glu release induced by veratridine in the absence of

external Ca2+; vinpocetine being the most potent [6, 7].

Also in the guinea pig in vivo phenytoin and carbamaze-

pine were less potent than vinpocetine to inhibit the

epileptic cortical activity induced by the convulsing agent,

pentylenetetrazole [8].

Vinpocetine is well tolerated in humans [9] and to our

knowledge no reports of motor disturbances caused by

M. Sitges (&) � B. I. Aldana � L. M. Chiu

Depto. de Biologıa Celular y Fisiologıa, Instituto de

Investigaciones Biomedicas, Universidad Nacional Autonoma de

Mexico, Apartado Postal 70228, Ciudad Universitaria,

04510 Mexico, D.F., Mexico

e-mail: sitges@biomedicas.unam.mx

V. Nekrassov

Division de Investigacion Basica y Aplicada, Instituto Nacional

de Rehabilitacion, SSA, Mexico, D.F., Mexico

123

Neurochem Res (2009) 34:470–479

DOI 10.1007/s11064-008-9805-7

vinpocetine exist. In contrast, several reports indicate that

phenytoin can induce dyskinesia [10–16] and some reports

have implicated carbamazepine in the production of

abnormal involuntary movements [17, 18].

Under conditions in which the energy supply surpasses

the energy demand, such as in models of experimental

epilepsy or ischemia, a massive release of striatal dopa-

mine accompanied by a decrease of its metabolites,

DOPAC and HVA takes place [19–24]. Interestingly

vinpocetine protects against neuronal damage induced by

cerebral ischemia [25–29], and increases the external

concentration of the DOPAC under baseline as well as

under depolarized conditions without changing dopamine

baseline release [30].

In an attempt to explore the possibility of a dopamine

involvement in the eventual motor disturbances caused by

the classic antiepileptic drugs, besides testing phenytoin

and carbamazepine effects under resting and veratridine-

depolarized conditions on dopamine release simultaneously

with their effects on Glu and Asp release, their effects on

the concentrations of dopamine and its main metabolite

(DOPAC) inside and outside striatal isolated nerve endings

were measured, and compared with the effects of vinp-

ocetine and of the MAO-I inhibitor, clorgyline.

Materials and Methods

Source of Materials

Drugs used in the experiments were acquired from the

following companies: vinpocetine (eburnamenine-14-car-

boxylic acid ethyl ester) was obtained either from Sigma

Chemical Co. (St. Louis, MO) or from Psicofarma S.A. de

C.V. (Mexico). Carbamazepine was obtained either from ICN

Biomedicals Inc. (Ohio) or from Psicofarma S.A. de C.V.

(Mexico). Phenytoin (5,5-diphenylhydantoin sodium salt)

was from Psicofarma S.A. de C.V. (Mexico). Veratridine,

n-methyl-n-propargyl-3-(2,4-dichloro-phenoxy)propylamine

(clorgyline) and 1-octanesulfonic acid were from Sigma

Chemical Co. (St. Louis, MO). The o-phthalaldehyde reagent

solution was from Pierce (Rockford, ILL). All other reagents

were of analytical grade.

Preparation of Synaptosomes

Dissected striata of 4 male Wistar rats (250–300 g) were

immediately placed in cold isotonic sucrose (1:10, w/v) and

homogenized (6 strokes at 2000 rpm, 0.15 mm pestle-

vessel clearance). The resulting suspensions were centri-

fuged at 1100g for 10 min, and supernatants obtained

from this centrifugation were centrifuged for another 20

min at 8200g. The resulting pellets containing striatal

synaptosomes were resuspended in standard Kreb’s Ringer

HEPES (KRH). The composition of the KRH is, in mM:

127 NaCl, 1.2 KH2PO4, 3.37 KCl, 1 CaCl2, 1.18 MgSO4,

20 HEPES and 5.6 mM dextrose, pH 7.4, bubbled with a

O2/CO2 mixture.

Release Experiments

Aliquots of striatal synaptosomes (450 ± 43 lg) suspended

in 500 ll of KRH were pre-incubated at 37�C for 5 min

before exposure to the experimental conditions to be

studied. Namely, carbamazepine (500 and 1500 lM),

phenytoin (range from 70 to 1500 lM), vinpocetine (15

lM), or clorgyline (0.1–10 lM), in the absence or in the

presence of 5 lM veratridine, and then incubated at 37�C

for 10 min. Incubation was stopped by centrifugation. The

supernatants resulting from this centrifugation containing

the released neurotransmitters were transferred to clean

vials and treated with an aliquot of a 0.1 M perchloric acid

(PCA)/0.1 mM EDTA solution, and stored at -40�C for

later analysis.

The resulting pellets were resuspended in 1 ml of the

PCA/EDTA solution pH 1.4 and vigorously vortex mixed.

These drastic conditions guarantee a complete discharge of

the synaptosomal content. The vortex mixed suspension

containing the disrupted synaptosomes was centrifuged.

The supernatants resulting from this centrifugation (con-

taining the neurotransmitters that were inside of each

synaptosomal sample) were also stored at -40�C for later

analysis. In order to standardize neurotransmitter release

per mg of synaptosomal protein, the resulting pellets were

suspended in 2 ml of a NaOH 5 mM solution and used for

protein determination. The samples containing the neuro-

transmitters released were injected into the HPLC system

within the 2 weeks after the experiment. Results are

expressed as the concentration of Glu (in nmol), Asp (in

nmol), dopamine (in pmol) or DOPAC (in pmol) released

or retained inside striatal synaptosomes per mg of synap-

tosomal protein.

Determination of the Concentrations of Dopamine and

DOPAC

Twenty microliters of sample suspended in 0.1 M PCA/0.1

mM EDTA were injected directly into a Waters HPLC

system for analysis. The HPLC system consists of a

delivery pump (model 600), a Rheodyne injector, an ana-

lytical column (resolve, C18, 150 9 3.9 mm internal

diameter, particle size 5 lm) controlled at 30�C, and an

electrochemical detector (model DECADE) with glassy

carbon used at a voltage of +0.8 V vs. a KCl (3 M) ref-

erence electrode (range 1 nA). A mobile phase composed

of 50 mM ortho-phosphoric acid/50 mM citric acid buffer,

Neurochem Res (2009) 34:470–479 471

123

pH 3.1 adjusted with KOH, containing 5% (v/v) methanol,

100 mg/l octanesulfonic acid and 20 mg/l EDTA, at a flow

rate of 1 ml/min, was applied for catecholamine elution.

Dopamine and DOPAC concentrations in the experimental

samples were calculated with calibration curves obtained

from the injection of increasing concentrations of the

external standard mixture (containing dopamine, DOPAC,

Glu and Asp) into the HPLC system.

Determination of the Concentrations of Glu and Asp

Ten microliters of sample suspended in 0.1 M PCA/0.1

mM EDTA was mixed with 20 ll of o-phthalaldehyde

reagent. After 120 s (strict time), a 10 ll aliquot of that

mixture was injected into the HPLC system. An analytical

column (Nova-pak C-18, 75 9 3.9 mm internal diameter,

particle size 10 lm) set at 25�C and a fluorescence detec-

tor set at 360 nm excitation wavelength and 450 nm

emission wave length were used. A lineal gradient elution

program performed over 30 min was applied for amino

acid elution: eluent A (30 mM sodium acetate buffer,

pH 6.8) from 100 to 50%, and eluent B (methanol) from

0 to 50%, at a flow rate of 1 ml/min. The concentrations of

Glu and Asp in the experimental samples were calculated

with calibration curves obtained from the injection of

increasing concentrations of the external standard mixture

after o-phthalaldehyde reagent derivatization into the

HPLC system.

Statistical Analysis

Paired Student’s t test was used for statistical evaluations.

From P \ 0.05 differences between data were considered

statistically significant. However most of the significant

differences between paired data obtained in parallel (i.e.

control and a specific condition) were below this value, as

indicated in some of the figures.

Results

Changes Induced by Carbamazepine, Phenytoin and

Vinpocetine on Glu, Asp and Dopamine Release in

Striatum Isolated Nerve Endings Incubated in the

Absence and Presence of Veratridine

When control neurotransmitter release values under resting

conditions (first line on Table 1) are compared with neu-

rotransmitter release values in the presence of the

antiepileptic drugs under resting conditions, a selective

increase exerted by carbamazepine and phenytoin on

dopamine baseline release becomes evident. Glu and Asp

baseline release remain unchanged by the presence of the

drugs tested.

The concentrations of glutamate, aspartate and dopa-

mine released for 10 min under resting conditions from

striatal synaptosomes (first line on Table 1) were markedly

increased by 5 lM veratridine (second line on Table 1). In

the presence of carbamazepine (1500 lM), phenytoin (200

lM) and vinpocetine (15 lM) the veratridine-induced

release of the excitatory amino acids was significantly

reduced (first two columns on Table 1). The veratridine-

induced release of dopamine also was reduced in the

presence of carbamazepine and vinpocetine at the above

concentrations, whereas phenytoin was paradoxically

unable to prevent the veratridine-induced increase in

dopamine release (third column on Table 1).

Table 1 Effect of AEDs on veratridine-evoked release of excitatory amino-acid neurotransmitters and dopamine in striatal synaptosomes

Glutamate Aspartate Dopamine

Control 16.0 ± 2.2 7.6 ± 1.3 9.1 ± 0.9

Veratridine 34.1 ± 2.8a 12.9 ± 1.0a 43.7 ± 7.6a

Carbamazepine 1500 lM 14.9 ± 2.3 6.5 ± 0.9 20.8 ± 4.5b

Carbamazepine 1500 lM + veratridine 15.4 ± 2.2c 5.9 ± 0.9c 30.2 ± 4.9a,c

Phenytoin 200 lM 14.6 ± 1.7 8.6 ± 1.6 19.6 ± 3.4b

Phenytoin 200 lM + veratridine 24.9 ± 2.0a,c 9.8 ± 1.1c 52.3 ± 5.5a,c

Vinpocetine 15 lM 13.7 ± 2.1 5.9 ± 0.7 8.1 ± 0.7

Vinpocetine 15 lM + veratridine 21.1 ± 2.1a,c 7.9 ± 0.6a,c 16.6 ± 0.9a,c

Amino acids are in nmol/mg and dopamine in pmol/mg synaptosomal protein. Results are the mean ± SEM values of 6 independent experiments

in parallela P \ 0.03 between neurotransmitter release in the absence and presence of veratridine under the indicated conditionb P \ 0.03 between the baseline release in the absence (control) and presence of the indicated AEDc P \ 0.03 between veratridine-induced release in the absence and presence of the indicated AED

472 Neurochem Res (2009) 34:470–479

123

Phenytoin, Clorgyline and Carbamazepine Effects

on Dopamine Distribution Inside and Outside

Synaptosomes Under Resting Conditions

In an attempt to get a better understanding of the selective

increase on dopamine baseline release exerted by phenyt-

oin and carbamazepine, their effects, at increasing

concentrations, on dopamine release and on the internal

concentrations of dopamine and its metabolite DOPAC

were measured, and compared with the effect of increasing

concentrations of clorgyline.

The progressive increase induced by phenytoin in the

range from 70 to 1500 lM on dopamine baseline release

and on the internal dopamine concentration is shown in

Fig. 1a, b, respectively. Similarly to phenytoin, increasing

concentrations of clorgyline (from 0.1 to 10 lM) progres-

sively increased both the external (Fig. 1c) as well as the

internal dopamine concentration (Fig. 1d). Carbamazepine

at high doses (500 and 1500 lM) also raised dopamine

baseline release dose dependently (Fig. 1e). However, in

contrast to phenytoin and clorgyline, the internal dopamine

concentration was lowered by carbamazepine (Fig. 1f).

Effect of Phenytoin, Clorgyline and Carbamazepine on

the Concentration of DOPAC Inside Synaptosomes

Under Resting Conditions

Oppositely to the raise in the internal dopamine concen-

tration, the increasing concentrations of phenytoin and

clorgyline progressively lowered the internal DOPAC

concentration under resting conditions (Fig. 2a, b). Car-

bamazepine in contrast failed in modifying internal

DOPAC significantly (Fig. 2c).

Figure 3 shows that the baseline release of dopamine

was unchanged by vinpocetine (15 lM), and that oppo-

sitely to phenytoin and clorgyline, vinpocetine decreased

internal dopamine and increased internal DOPAC.

We just tested vinpocetine at one concentration here

because we have already characterized the dose response

effects of vinpocetine on the internal and external

[clorgyline] (µM)

[DA

] re

leas

ed

10

20

30P = 0.002

P = 0.0002

P = 0.02

[DA

] in

sid

e

210

280

350

420

[clorgyline] (µM)

P = 0.00001

P = 0.004P = 0.0001

0 500 1000 1500

[DA

] in

sid

e

210

280

350

420

[carbamazepine] (µM)

P = 0.049P = 0.02

[carbamazepine] (µM) 0 500 1000 1500

[DA

] re

leas

ed

10

20

30P = 0.03

P = 0.001

0 250 500 1500

[DA

] in

sid

e

210

280

350

420

[phenytoin] (µM)

P = 0.03P = 0.001

P = 0.0001

P = 0.03

[phenytoin] (µM)0 250 500 1500

[DA

] re

leas

ed

10

20

30 P = 0.01P = 0.01

P = 0.0003

P = 0.01(a) (b)

(d)(c)

(e) (f)

0 3 6 9 0 3 6 9

Fig. 1 Changes induced by

phenytoin, clorgyline and

carbamazepine at increasing

concentrations in dopamine

distribution. Striatal

synaptosomes were incubated

(37�C) for 10 min in: KRH

without (zero) or containing

increasing concentrations of:

phenytoin (upper graphs),

clorgyline (middle graphs) or

carbamazepine (bottom graphs).

Incubation was stopped by

centrifugation. Samples

containing dopamine released

and retained were prepared for

HPLC analysis (see section

‘‘Release Experiments’’ for

details) and the pellets used for

protein determination.

Dopamine (DA) released (left

columns) or retained (right

columns) is in pmol/mg of

synaptosomal protein. Results

are the mean ± SEM values of

at least 4 experiments in

independent preparations for

each point. The statistical

significance (P) between a

particular drug concentration

and its respective control is

indicated

Neurochem Res (2009) 34:470–479 473

123

distribution of dopamine and DOPAC in striatal synapto-

somes previously [30].

Comparison of Clorgyline, Phenytoin, Carbamazepine

and Vinpocetine Effects on External DOPAC Under

Resting Conditions

In nerve endings incubated for 10 min in the presence of

clorgyline the external DOPAC concentration was signifi-

cantly lowered even from the lowest clorgyline

concentration tested. The external DOPAC concentration

was unchanged by phenytoin whereas carbamazepine

slightly lowered external DOPAC and vinpocetine

increased it (Table 2).

The control value shown in Table 2, for practical reasons

is the average of all the independent experiments that were

included in the table. However statistical differences were

calculated between the external DOPAC concentration

values obtained in parallel in the absence and in the pres-

ence of the particular concentration of the specified drug.

Comparison of Clorgyline, Phenytoin and

Carbamazepine Effects on the Net Release of

Neurotransmitters Induced by Veratridine

Glu and Asp baseline release under resting conditions was

unchanged by clorgyline. For instance in the presence of

0.1 lM clorgyline 16.3 ± 1.5 nmol of Glu and 7.1 ± 0.6

nmol of Asp per mg of striatal synaptosomal protein were

released. These values are very similar to the control

baseline release values shown in Table 1.

[DO

PA

C] i

nsid

e

30

60

90

[clorgyline] (µM)

P = 0.001 P = 0.007

0 500 1000 1500

[DO

PA

C] i

nsid

e

30

60

90

[carbamazepine] (µM)

0 250 500 1500

[DO

PA

C] i

nsid

e

30

60

90

[phenytoin] (µM)

P = 0.02P = 0.01

P = 0.01

P = 0.005

(a)

(b)

(c)

P = 0.00002

0 3 6 9

Fig. 2 Changes induced by phenytoin, clorgyline and carbamazepine

at increasing concentrations in internal DOPAC. Striatal synapto-

somes were treated as in Fig. 1

15

[DA

] rel

ease

d

10

20

30

[DA

] ins

ide

210

280

350

420

[DO

PA

C] i

nsid

e

60

90

120

P = 0.046

[vinpocetine] (µM)

(a)

(b)

(c)

P = 0.04

0

150

150

Fig. 3 Vinpocetine-induced changes in dopamine release and in the

internal catecholamine concentrations. Striatal synaptosomes were

incubated for 10 min in KRH without (zero) or containing 15 lM

vinpocetine and treated as in the above figures. Dopamine (DA) and

DOPAC concentrations are in pmol/mg of synaptosomal protein.

Results are the mean ± SEM values of five experiments in

independent preparations

474 Neurochem Res (2009) 34:470–479

123

Phenytoin at higher concentrations than the concentra-

tion used in the first set of experiments shown in table 1

(200 lM) also failed in modifying Glu and Asp baseline

release. For instance, in the presence of 500 and 1500 lM

phenytoin 14.7 ± 2.6 and 16.8 ± 2.8 nmol/mg of Glu per

mg of striatal synaptosomal protein were released,

respectively. The baseline release of the excitatory amino

acid neurotransmitters also was unchanged by 500 lM

carbamazepine.

For comparing the effect of the MAO-I clorgyline with

the effect of the classic antiepileptic drugs on the veratri-

dine-evoked release of the excitatory amino acid

neurotransmitters and dopamine, the net effect of veratri-

dine on neurotransmitter release was calculated. Because

although Glu and Asp baseline release is unchanged by

clorgyline, phenytoin and carbamazepine, in the same

range of concentrations they all increased dopamine

baseline release (as shown in Fig. 1). The net veratridine-

induced neurotransmitter release refers to the neurotrans-

mitter released in the presence of veratridine under a

specific experimental condition minus the release in the

absence of veratridine under the same experimental con-

dition. Figure 4a shows that whereas clorgyline (0.1 lM)

failed in modifying the net veratridine-induced release of

Glu, phenytoin at increasing concentrations (200, 500, and

1500 lM) progressively inhibited the net release of Glu

induced by veratridine, and carbamazepine (1500 lM)

practically abolished it. The same pattern was observed for

Asp, the other excitatory amino acid neurotransmitter

tested (Fig. 4b). Interestingly, the release of the neuro-

transmitter dopamine triggered by activation of Na+

channels with veratridine was dramatically increased by

clorgyline, insensitive to 200 lM phenytoin and facilitated

by the higher phenytoin concentrations tested (500 and

1500 lM). In contrast, the release of dopamine triggered by

veratridine was markedly reduced by carbamazepine

(Fig. 4c).

Discussion

The present study compares the effects of several drugs on

the simultaneous release of Glu, Asp and dopamine from

striatal isolated nerve endings under resting conditions and

under conditions in which sodium channel permeability

was increased with veratridine.

Present results show that except the MAO-I clorgyline,

all the compounds with anticonvulsant capabilities, namely

carbamazepine, phenytoin and vinpocetine reduce the

veratridine-induced release of Glu and Asp. This sensitivity

of the veratridine-induced increase in excitatory aminoacid

neurotransmitter release is in agreement with previous

studies showing that phenytoin and carbamazepine dis-

place [3H]batrachotoxin binding to Na+ channels in

cerebral membranes and brain isolated nerve endings

[31–35], and with studies showing that vinpocetine

inhibits the tetrodotoxin sensitive fraction of the rise in

Na+ induced by veratridine and by the convulsing agent

4-amonipyridine in cerebral isolated nerve endings

[36–38]. The inhibition of Glu and Asp release to veratri-

dine by those compounds is also in line with the idea that

antiepileptic drugs suppress the abnormal neuronal excit-

ability associated with seizures by means of Na+ channel

blockade. However, the increase on dopamine release

induced by carbamazepine and phenytoin under resting

conditions, within the same range of concentrations that

inhibited the release of the excitatory amino acid neuro-

transmitters triggered by Na+ channel activation, indicates

Table 2 Effect of drugs on external DOPAC in striatal synaptosomes

Drug-induced change External [DOPAC] Statistical significance (P) Number of experiments

Control 274 ± 16

Clorgyline 0.1 lM ; 214 ± 13 0.0004 n (5)

Clorgyline 1 lM ; 203 ± 10 0.005 n (4)

Clorgyline 10 lM ; 213 ± 18 0.004 n (4)

Phenytoin 70 lM = 277 ± 12 0.90 n (6)

Phenytoin 200 lM = 269 ± 15 0.80 n (6)

Phenytoin 500 lM = 277 ± 16 0.47 n (4)

Phenytoin 1500 lM = 281 ± 23 0.44 n (4)

Carbamazepine 500 lM ; 249 ± 17 0.02 n (6)

Carbamazepine 1500 lM ; 246 ± 9 0.002 n (6)

Vinpocetine 15 lM : 294 ± 8 0.04 n (5)

External DOPAC concentrations are in pmol/mg of synaptosomal protein. P, statistical difference between external [DOPAC] obtained in the

absence (control) and in the presence of the indicated concentration of the drug. Results are the mean ± SEM values of the number (n) of

independent experiments in parallel. The control external [DOPAC] value is the average of 18 independent experiments

Neurochem Res (2009) 34:470–479 475

123

an additional action of those antiepileptic drugs. Particu-

larly the phenytoin induced facilitation of dopamine release

observed under both resting and veratridine-depolarized

conditions cannot be explained by a reduction of presyn-

aptic Na+ channels permeability.

Present results were obtained in striatal isolated nerve

endings. This preparation mainly includes glutamate

releasing nerve endings from the cortico-striatal pathway

and dopamine releasing nerve endings from the nigro-

striatal pathway, but may also contain some microglial

elements. In this preparation both, phenytoin and carbam-

azepine increased the release of the monoamine

neurotransmitter dopamine. In the central nervous system

monoamines regulate microglial activation via extrasy-

naptic communication phenomena [39]. In addition, in

various physiological states as well as pathological states

including epilepsy microglial activation takes place [40–

42]. Therefore, it is possible that the increase in dopamine

release induced by the above antiepileptic drugs could

contribute to some of their adverse secondary effects par-

ticularly in epileptic patients.

Monoamino-oxidase (MAO) is the enzyme that converts

dopamine to DOPAC [43]. The high potency of the selective

MAO-A inhibitor clorgyline to inhibit dopamine metabo-

lism, suggest a primary role of this type of MAO in

dopamine catabolism. The clorgyline-induced dopamine

increase and DOPAC decrease shown here is in line with

previous findings using microdialysis [44] and also with our

previous work in striatal synaptosomes [30]. The increase in

dopamine induced by phenytoin was also accompanied by a

decrease in internal DOPAC formation suggesting that the

dose-dependent increase in dopamine baseline release

induced by phenytoin also involves an inhibition of

dopamine metabolism. The reduced external DOPAC con-

centration produced by clorgyline is explained by the

complete inhibition of dopamine metabolism with con-

comitant decrease in DOPAC formation. Phenytoin failure

to change the external concentration of the dopamine

metabolite is probably explained by the much lower efficacy

of phenytoin than clorgyline to stop dopamine metabolism.

Oppositely to clorgyline, vinpocetine increased external

DOPAC. The mechanism responsible of this increase in

DOPAC exerted by vinpocetine was previously character-

ized by us [30], and does not involve MAO activation, but

rather is likely to be due to an impairment of dopamine

vesicular storage.

The amount of dopamine release under resting conditions

is likely to depend on the concentration of dopamine in the

cytoplasm extravesicular compartment [30]. Therefore, the

impairment of cytoplasm extravesicular dopamine degra-

dation induced by phenytoin and by clorgyline that results in

a rise in the internal dopamine concentration may underlie

the increase in dopamine baseline release induced by those

drugs. Carbamazepine, like phenytoin, also increased

dopamine baseline release. However, the mechanism

underlying the carbamazepine-induced increase in dopa-

mine baseline release is inconsistent with a MAO inhibition.

Because oppositely to phenytoin (and clorgyline) that

increased the internal dopamine concentration, carbamaze-

pine decreased the internal dopamine concentration. The

Net

ver

atri

din

e-in

du

ced

neu

rotr

ansm

itte

r re

leas

ed

op

amin

e

0

15

30

45

60

105

**

**

Glu

tam

ate

5

10

15

20

**

*

Asp

arta

te

0

4

8

**

*

*

*

(a)

(b)

(c)

Fig. 4 Changes induced by clorgyline, phenytoin and carbamazepine

on the net response to veratridine. Striatal synaptosomes were

incubated (37�C) for 10 min in: KRH without or containing

veratridine in the absence or presence of phenytoin, clorgyline or

carbamazepine at the indicated concentrations. Incubation was

stopped by centrifugation. The samples containing the released and

retained neurotransmitters, namely glutamate (a), aspartate (b) or

dopamine (c), were prepared for HPLC analysis and the pellets used

for protein determination. Net veratridine-induced release refers to the

concentration of a specific neurotransmitter released by veratridine

for 10 min in the presence of: 0.1 lM clorgyline (clorg.), phenytoin

(200, 500 or 1500 lM) or carbamazepine (1500 lM) minus the

neurotransmitter released under the respective baseline condition for

10 min (i.e. 0.1 lM clorgyline, 200 lM phenytoin, etcetera.). For (a)

and (b) results are in nmol and for (c) in pmol per mg of striatal

synaptosomal protein. Results are the mean ± SEM values of at least

4 experiments in independent preparations for each point. * P B 0.01,

between control (black bar) and the indicated experimental condition

(grey bars)

476 Neurochem Res (2009) 34:470–479

123

reduction produced by carbamazepine in the internal dopa-

mine concentration, i.e. in the MAO substrate, also may

explain the slight decrease in the amount of external DOPAC

observed in carbamazepine exposed nerve endings. Car-

bamazepine-induced increase in dopamine baseline release

at expense of internal dopamine may involve mechanisms

such as inhibition of the dopamine transporter or carbam-

azepine hetero-exchange with internal dopamine, but not

inhibition of dopamine metabolism. It is important to realize

that high K+ depolarization increases dopamine baseline

release about threefold [45], and the drugs tested here in

about the same extent. Moreover, on the basis of evidence

indicating the presence of presynaptic autoreceptors that

enable catecholamines, including dopamine, to reduce their

own release via feedback modulation [46, 47], it is possible

that the rise on external dopamine induced by depolarization

or by some of the drugs tested here under the present

experimental conditions is underestimated.

Another difference between the two classic antiepileptic

drugs is that phenytoin failed to inhibit and at higher

concentrations even facilitated the veratridine-induced

release of dopamine. Although, the possibility of a cross-

talk between glutamatergic and dopaminergic nerve

endings via presynaptic hetero-receptors underlying this

particular effect of phenytoin cannot be discarded, it is

unlikely. Because carbamazepine, that like phenytoin also

increases dopamine baseline release and inhibits Glu

release to veratridine, does not facilitate the veratridine

induced release of dopamine, and rather decreased it like

vinpocetine. The phenytoin facilitation of veratridine-

induced dopamine release found here also was observed in

microdialysis experiments of the hippocampus in vivo [48].

Our findings that carbamazepine inhibits the release of all

the neurotransmitters tested including dopamine in striatal

isolated nerve endings also are in line with the sensitivity to

carbamazepine of veratrine-induced release of [3H]dopa-

mine in rat cortical slices preloaded with the labeled

neurotransmitter [49], but contrast with the failure of car-

bamazepine to inhibit dopamine release to veratridine in

the hippocampus [48]. One possible explanation of this

failure could be the high veratridine concentration used,

that in neurons of the hippocampal formation in culture

was shown to induce leakage of the cytoplasmic protein

lactate dehydrogenase [50].

Glu followed by Asp are by far the most abundant neu-

rotransmitters even in cerebral nerve endings arriving to the

striatum [45]. Thus, the inhibition of the veratridine induced

release of those excitatory amino acid neurotransmitters

produced by the anticonvulsants carbamazepine, phenytoin

and vinpocetine (and not by clorgyline) might be of par-

ticular importance to stop the paroxysmal neuronal activity.

Lamotrigine is another antiepileptic drug that antagonizes

Glu release induced by activation of cerebral Na+ channels

with a similar potency to carbamazepine and phenytoin [7].

Interestingly, in the presence of a monoamine agonist,

lamotrigine increased its potency to antagonize spinal Na+

channels [51]. Since microglial activation modifies the

accumulation and diffusion parameters of brain neuroactive

substances [40], it is possible that the monoamine-induced

microglial activation produced in the central nervous sys-

tem by cross talk non-synaptic mechanisms [52, 53], further

increases the potency of antiepileptic drugs on Na+ chan-

nels in the epileptic tissue, where inflammatory pathways

are also chronically activated [41].

Our findings that phenytoin failed to inhibit the release

of dopamine triggered by veratridine at the same concen-

trations and under the same experimental conditions that

reduced the veratridine induced release of Glu and Asp

lead, however, to speculate whether different populations

of Na+ channels control the release of the excitatory amino

acid neurotransmitters and dopamine. Nonetheless, in

addition to this antagonism on presynaptic Na+ channels

controlling Glu and Asp release, the classic antiepileptic

drugs, carbamazepine and phenytoin produced an addi-

tional effect on dopamine release and/or metabolism that

may possibly contribute to some of their adverse secondary

effects, such as the dyskinesias and paradoxical seizures

observed in some phenytoin treated patients or the abnor-

mal involuntary movements less frequently caused by

carbamazepine [10–18, 54, 55]. Supporting this idea the

NMDA receptor antagonist MK-801, that has anticonvul-

sant effects [56, 57] and causes disturbances in motor

coordination [58], inhibited the veratridine-evoked release

of Glu and Asp and selectively increased dopamine base-

line release in striatal synaptosomes [45].

Like carbamazepine, phenytoin and MK-801, vinpoce-

tine inhibited veratridine-evoked Glu and Asp release, and

also like carbamazepine and MK-801, vinpocetine inhib-

ited the veratridine-evoked release of dopamine,

confirming its antagonistic action on presynaptic Na+

channels. But oppositely to carbamazepine, phenytoin and

MK-801, vinpocetine did not increase dopamine baseline

release. This failure of vinpocetine to increase dopamine

baseline levels along with its higher potency and efficacy

than several commonly used antiepileptic drugs to prevent

Glu release induced by activation of voltage sensitive

presynaptic channels in cerebral isolated nerve endings

[59] and to prevent seizures in animal models of epilepsy

[8, 60, 61] further supports the vinpocetine potential as an

alternative antiepileptic drug.

Acknowledgments The authors thank Araceli Guarneros for her

excellent technical assistance. This work was partially supported by

grants 225008 from PAPIIT and D-48695 from SEP-CONACYT.

Blanca I. Aldana Garcıa scholarship was also supported by grant

225008 from PAPIIT.

Neurochem Res (2009) 34:470–479 477

123

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